Tyrosine-phosphatase-related protein

ABSTRACT

The present invention relates to a tyrosine-phosphatase-related protein, a DNA which codes for such a protein and a process for producing such a protein. In addition, the invention concerns the use of the DNA and protein and antibodies directed against the protein.

The present invention relates to a tyrosine-phosphatase-related protein, a DNA which codes for such a protein and a process for producing such a protein. In addition, the invention concerns the use of the DNA and protein and antibodies directed against the protein.

Tyrosine kinase and tyrosine phosphatase are enzymes which have opposite effects. Tyrosine kinase effects the phosphorylation of certain tyrosine residues in proteins, whereas tyrosine phosphatase reverses this phosphorylation again. Both enzymes play an important part for the signal transduction, the control of cell growth and cell differentiation.

Diseases are known which are based on disturbances occurring in Tell differentiation. One of these diseases is myotubular myopathy. It is an X-chromosomal-connected disease which is accompanied by altered muscle cells. This disease manifests itself in general muscle weakness, particularly spontaneous movements are hardly possible. Likewise, the respiration of newborns is strongly confined. This frequently leads to premature death However, the causes of disturbed cell differentiation in the case of myotubular myopathy are not known.

Therefore, it is the object of the present invention to provide a product by which it is possible to investigate the cause of cell differentiation disturbances, particularly in the case of myotubular myopathy, and optionally treat them. According to the invention this is achieved by the subject matters defined in the claims.

Thus, the subject matter of the present invention relates to a tyrosine-phosphatase-related protein, the protein comprising the amino acid sequence of FIG. 1 or an amino acid sequence differing therefrom by one or several amino acids.

The present invention is based on the applicant's finding that a protein exists in animals, particularly mammals, more particularly human beings, which has homologies with known tyrosine phosphatases and also tyrosine phosphatase activity but differs from known tyrosine phosphatases on the DNA level by hybridization under normal conditions. Such a protein has the amino acid sequence of FIG. 1 or an amino acid sequence differing therefrom by one or several amino acids. The applicant has also discovered that the protein is important for cell differentiation. He has found that, in its shortened form and mutated form, respectively, i.e. without or only with restricted tyrosine phosphatase activity, this protein disturbs the differentiation of cells, particularly muscle cells, and more particularly leads to the formation of myotubular myopathy.

The above protein is referred to as “tyrosine-related protein” (TVP) in the present invention.

A further subject matter of the present invention relates to a nucleic acid coding for (TVP). It may be an RNA or a DNA. The latter may be e.g. a genomic DNA or a cDNA. Preferred is a DNA comprising the following:

(a) the DNA of FIG. 1 or a DNA differing therefrom by one or several base pairs,

(b) a DNA hybridizing with the DNA of (a), or

(c) a DNA related to the DNA of (a) or (b) via the degenerated genetic code.

The expression “hybridizing DNA” refers to a DNA which hybridizes with a DNA of (a) under normal conditions, particularly at 20° C. below the melting point of the DNA.

The DNA of FIG. 1 was deposited wit h the DSM (Deutsche Sammlung von Mikroorganismen und Zellkulturen [German-type collection of micro-organisms and cell cultures]) as hp6 under DSM 10558 on Mar. 4, 1996.

A DNA according to the invention is described below in the form of a cDNA. It is exemplary for every DNA falling under the present invention.

For preparing a cDNA according to the invention, it is favorable to use as a basis a cosmid library which comprises the region Xq28 of the human genome. Such a cosmid library is e.g. the Xq28-specific cosmid library (cf. Kioschis, P. et al., Cytogenet. Cell. Genet. 58, (1991), 2070) which was prepared from the cell hybrid QIZ (cf. Warren, S. T. et al., Proc. Natl. Acad. Scif U.S.A. 87 (1990), 3856-3860). The cosmid clones Qc8D11, Qc3F12 and Qc12G11 thereof (cf. Kioschis, P. et al., Cytogenet. Cell. Genet. 58, (1991), 2070; Kioschis, P. et al., Genomics 33, (1996) in print) are used and subjected to DNA selection (cf. Korn, B. et al., Mol. Genet. 4 (1992), 235-242) s o as to obtain the cDNA fragment 79g1P5. It is used for hybridizing a cDNA library of human placenta (e.g. STRATAGENE, catalog No. 936203). A cDNA according to the invention is obtained.

A cDNA according to the invention may be present in a vector and expression vector, respectively. A person skilled in the art is familiar with examples thereof. In the case of an expression vector for E. coli these are e.g. pGEMEX, pUC derivatives, pGEX-2T, pET3b and pQE-8, the latter being preferred. For the expression in yeast, e.g. pY100 and Ycpad1 have to be mentioned while e.g. pkCR, pEFBOS, cDM8 and pCEV4 have to be indicated for the expression in animal cells . The baculovirus expression vector pAcSGHisNT-A is especially suitable for the expression in insect cells.

The person skilled in the art is familiar with suitable cells to express a cDNA according to the invention, which is present in an expression vector. Examples of such cells comprise the E. coli strains RB101, DH1, x1776, JM101, JM109, BL21 and SG13009, the latter being preferred, the yeast strain saccharomyces cerevisiae and the animal cells L, 3T3, FM3A, CHO, COS, Vero and HeLa as well as the insect cells sf9.

The person skilled in the art knows in which way a DNA according to the invention has to be inserted in an expression vector. He is also familiar with the fact that this DNA can be inserted in combination with a DNA coding for another protein and peptide, respectively, so that the cDNA according to the invention can be expressed in the form of a fusion protein.

In addition, the person skilled in the art knows conditions of culturing transformed cells and transfected cells, respectively. He is also familiar with processes of isolating and purifying the protein expressed by the cDNA according to the invention. Thus, such a protein, which may also be a fusion protein, is also a subject matter of the present invention.

A further subject matter of the present invention relates to an antibody directed against an above protein and fusion protein, respectively. Such an antibody can be prepared by common methods. It may be polyclonal and monoclonal, respectively. For its preparation it is favorable to immunize animals—particularly rabbits or chickens for a polyclonal antibody and mice for a monoclonal antibody—with an above (fusion) protein or with fragments thereof. Further “boosters” of the animals can be effected with the same (fusion) protein or with fragments thereof. The polyclonal antibody may then be obtained from the animal serum and egg yolk, respectively. For the preparation of the monoclonal antibody, animal spleen cells are fused with myeloma cells.

The present invention enables to investigate the causes of cell differentiation disturbances, particularly in the case of muscle cells, and more particularly in the case of myotubular myopathy. By means of a nucleic acid according to the invention, particularly a DNA, and primers derived therefrom, it can be determined in mammals, particularly human beings, whether they contain and/or express a gene which codes, within the above sense, for a shortened (TVP) and mutated (TVP), respectively. For this purpose, a person skilled in the art will carry out common methods such as reverse transcription, PCR reaction, hybridization and sequencing. A kit which contains an above nucleic acid, particularly DNA, and/or primers derived therefrom as well as carriers and conventional auxiliary agents, is also provided according to the invention.

Furthermore, the present invention is suited to take therapeutic measures in the case of cell differentiation disturbances, particularly in the case of muscle cells and more particularly in the case of myotubular myopathy. A (TVP) according to the invention can be inserted in mammals, particularly human beings. For this purpose, it may be favorable to couple (TVP) to a protein which is not considered foreign by the respective body, e.g. transferrin or BSA. A nucleic acid according to the invention, particularly a DNA, can also be inserted and expressed in mammals, particularly human beings. For this purpose, it may be favorable to have the expression of the nucleic acid according to the invention be controlled by a tissue-specific promoter, particularly a muscle-specific promoter. The expression of (TVP) can be controlled and regulated by an antibody according to the invention.

Thus, the present invention represents a great contribution to the diagnostic and therapeutic detection of disturbances of cell differentiation, particularly in the case of muscle cells and more particularly in the case of myotubular myopathy. In this connection, the diagnostic detection cannot only be made postnatally but also prenatally already.

BRIEF DESCRIPTION OF THE DRAWING

FIGS. 1A-1E shows the base sequence and the amino acid sequence, derived therefrom, of a (TVP) according to the invention.

The present invention is explained by the below examples.

Example 1 Preparation and Purification of a (TVP) According to the Invention

For preparing a (TVP) according to the invention, the DNA of FIG. 1 was used as a template. A PCR method was carried out. The following primer pair was used:

MTM-F: 5′-CAGGGATCCGATGGCAGCCGAGCAGCCTGGCAAC-3′ and MTM-RR: 5′-GGGGGATCCTCAGAAGTGAGTTTGCACATGGGG-3′

The PCR batch and PCR conditions were as follows:

PCR batch

Template DNA (FIG. 1): 1 μl=1 ng

Pfu polymerase 10x buffer: 10 μl=1 x

DMSO: 10 μl=10 %

dNTP's: 1 μl=200 μM each

oligonucleotides, 1.5 μl each: 3 μl=150 ng each

H₂O bidist.: ad 99 μl

PCR conditions

92° C.—5 min.

addition of 1 μl Pfu polymerase (Stratagene)=2.5 units

addition of paraffin

PCR 92° C. 1 min. 58° C. 1 min. 1 cycle 72° C. 10 min.  92° C. 1 min. 58° C. 1 min. 39 cycles  72° C. 2 min. 72° C. 10 min.  1 cycle

The amplified DNA was cleaved by Bam HI each and inserted in the expression vector pQE8 (Diagen company) cleaved by Bam HI. The expression plasmid pQ/TVP was obtained. Such a plasmid codes for a fusion protein comprising 6 histidine residues (N terminus partner) and the (TVP) of FIG. 1 according to the invention (C terminus partner). pQ/TVP was used for transforming E. coli SG 13009 (cf. Gottesman, S. et al., J. Bacteriol. 148, (1981), 265-273). The bacteria were cultured in an LB broth with 100 μg/ml ampicillin and 25 μg/ml kanamycin and induced with 60 μM isopropyl-β-D-thiogalactopyranoside (IPTG) for 4 h. Lysis of the bacteria was achieved by the addition of 6 M guanidine hydrochloride. Thereafter, chromatography (Ni-NTA resin) was carried out with the lysate in the presence of 8 M urea in accordance with the instructions from the manufacturer (Diagen company) of the chromatography material. The bound fusion protein was eluted in a buffer having a pH of 3.5. After its neutralization, the fusion protein was subjected to 18 % SDS polyacrylamide gel electrophoresis and stained with coomassie blue (cf. Thomas, J. O. and Kornberg, R. D., J. Mol. Biol. 149 (1975), 709-733).

It showed that a (fusion) protein according to the invention can be prepared in highly pure form.

Example 2 Preparation and Detection of an Antibody According to the Invention

A fusion protein of Example 1 according to the invention was subjected to 18 SDS polyacrylamide gel electrophoresis. After staining the gel with 4 M sodium acetate, an about 205 kD band was cut out of the gel and incubated in phosphate-buffered common salt solution. Gel pieces were sedimented before the protein concentration of the supernatant was determined by SDS polyacrylamide gel electrophoresis which was followed by coomassie blue staining. Animals were immunized with the gel-purified fusion protein as follows:

Immunization Protocol for Polyclonal Antibodies in Rabbits

35 μg of gel-purified fusion protein in 0.7 ml PBS and 0.7 ml of complete Freund's adjuvant and incomplete Freund's adjuvant, respectively, were used per immunization:

Day 0: 1^(st) immunization (complete Freund's adjuvant)

Day 14: 2^(nd) immunization (incomplete Freund's adjuvant; icFA)

Day 28: 3^(rd) immunization (icFA)

Day 56: 4^(th) immunization (icFA)

Day 80: bleeding to death.

The rabbit serum was tested in an immunoblot. For this purpose, a fusion protein of Example 1 according to the invention was subjected to SDS polyacrylamide gel electrophoresis and transferred to a nitrocellulose filter (cf. Khyse-Andersen, J., J. Biochem. Biophys. Meth. 10 (1984), 203-209). The Western blot analysis was carried out as described in Bock, C.-T. et al., Virus Genes 8, (1994), 215-229. For this purpose, the nitrocellulose filter was incubated with a first antibody at 37° C. for one hour. This antibody was the rabbit serum (1:10000 in PBS). After several wash steps using PBS, the nitrocellulose filter was incubated with a second antibody. This antibody was an alkaline phosphatase-coupled monoclonal goat anti-rabbit IgG antibody (Dianova company) (1:5000) in PBS. 30 minutes of incubation at 37° C. were followed by several wash steps using PBS and subsequently by the alkaline phosphatase detection reaction with developer solution (36 μM 5′-bromo-4-chloro-3-indolylphosphate, 400 μM nitro blue tetrazolium, 100 mM Tris-HCl, pH 9.5, 100 mM NaCl, 5 mM MgCl₂) at room temperature until bands were visible.

It showed that polyclonal antibodies according to the invention can be prepared.

Immunization Protocol for Polyclonal Antibodies in Chickens

40 μg of gel-purified fusion protein in 0.8 ml PBS and 0.8 ml of complete Freund's adjuvant and incomplete Freund's adjuvant, respectively, were used per immunization.

Day 0: 1^(st) immunization (complete Freund's adjuvant)

Day 28: 2^(nd) immunization (incomplete Freund's adjuvant; icFA)

Day 50: 3^(rd) immunization (icFA)

Antibodies were extracted from egg yolk and tested in a Western blot. Polyclonal antibodies according to the invention were detected.

Immunization Protocol for Monoclonal Antibodies in Mice

12 μg of gel-purified fusion protein in 0.25 ml PBS and 0.25 ml of complete Freund's adjuvant and incomplete Freund's adjuvant, respectively, were used per immunization. The fusion protein was dissolved in 0.5 ml (without adjuvant) in the 4^(th) immunization.

Day 0: 1^(st) immunization (complete Freund's adjuvant)

Day 28: 2^(nd) immunization (incomplete Freund's adjuvant; icFA)

Day 56: 3^(rd) immunization (icFA)

Day 84: 4^(th) immunization (PBS)

Day 87: fusion.

Supernatants of hybridomas were tested in a Western blot. Monoclonal antibodies according to the invention were detected.

4 1 3431 DNA German-type microorganism & cell cul. CDS (1)..(1863) 1 gca gcc gag cag cct ggc aac ggc ggt ggc gcc cgg agc ccg aga gtt 48 Ala Ala Glu Gln Pro Gly Asn Gly Gly Gly Ala Arg Ser Pro Arg Val 1 5 10 15 tcc agg atg gct tct gca tca act tct aaa tat aat tca cac tcc ttg 96 Ser Arg Met Ala Ser Ala Ser Thr Ser Lys Tyr Asn Ser His Ser Leu 20 25 30 gag aat gag tct att aag agg acg tct cga gat gga gtc aat cga gat 144 Glu Asn Glu Ser Ile Lys Arg Thr Ser Arg Asp Gly Val Asn Arg Asp 35 40 45 ctc act gag gct gtt cct cga ctt cca gga gaa aca cta atc act gac 192 Leu Thr Glu Ala Val Pro Arg Leu Pro Gly Glu Thr Leu Ile Thr Asp 50 55 60 aaa gaa gtt att tac ata tgt cct ttc aat ggc ccc att aag gga aga 240 Lys Glu Val Ile Tyr Ile Cys Pro Phe Asn Gly Pro Ile Lys Gly Arg 65 70 75 80 gtt tac atc aca aat tat cgt ctt tat tta aga agt ttg gaa acg gat 288 Val Tyr Ile Thr Asn Tyr Arg Leu Tyr Leu Arg Ser Leu Glu Thr Asp 85 90 95 tct tct cta ata ctt gat gtt cct ctg ggt gtg atc tcg aga att gaa 336 Ser Ser Leu Ile Leu Asp Val Pro Leu Gly Val Ile Ser Arg Ile Glu 100 105 110 aaa atg gga ggc gcg aca agt aga gga gaa aat tcc tat ggt cta gat 384 Lys Met Gly Gly Ala Thr Ser Arg Gly Glu Asn Ser Tyr Gly Leu Asp 115 120 125 att act tgt aaa gac atg aga aac ctg agg ttc gct ttg aaa cag gaa 432 Ile Thr Cys Lys Asp Met Arg Asn Leu Arg Phe Ala Leu Lys Gln Glu 130 135 140 ggc cac agc aga aga gat atg ttt gag atc ctc acg aga tac gcg ttt 480 Gly His Ser Arg Arg Asp Met Phe Glu Ile Leu Thr Arg Tyr Ala Phe 145 150 155 160 ccc ctg gct cac agt ctg cca tta ttt gca ttt tta aat gaa gaa aag 528 Pro Leu Ala His Ser Leu Pro Leu Phe Ala Phe Leu Asn Glu Glu Lys 165 170 175 ttt aac gtg gat gga tgg aca gtt tac aat cca gtg gaa gaa tac agg 576 Phe Asn Val Asp Gly Trp Thr Val Tyr Asn Pro Val Glu Glu Tyr Arg 180 185 190 agg cag ggc ttg ccc aat cac cat tgg aga ata act ttt att aat aag 624 Arg Gln Gly Leu Pro Asn His His Trp Arg Ile Thr Phe Ile Asn Lys 195 200 205 tgc tat gag ctc tgt gac act tac cct gct ctt ttg gtg gtt ccg tat 672 Cys Tyr Glu Leu Cys Asp Thr Tyr Pro Ala Leu Leu Val Val Pro Tyr 210 215 220 cgt gcc tca gat gat gac ctc cgg aga gtt gca act ttt agg tcc cga 720 Arg Ala Ser Asp Asp Asp Leu Arg Arg Val Ala Thr Phe Arg Ser Arg 225 230 235 240 aat cga att cca gtg ctg tca tgg att cat cca gaa aat aag acg gtc 768 Asn Arg Ile Pro Val Leu Ser Trp Ile His Pro Glu Asn Lys Thr Val 245 250 255 att gtg cgt tgc agt cag cct ctt gtc ggt atg agt ggg aaa cga aat 816 Ile Val Arg Cys Ser Gln Pro Leu Val Gly Met Ser Gly Lys Arg Asn 260 265 270 aaa gat gat gag aaa tat ctc gat gtt atc agg gag act aat aaa caa 864 Lys Asp Asp Glu Lys Tyr Leu Asp Val Ile Arg Glu Thr Asn Lys Gln 275 280 285 att tct aaa ctc acc att tat gat gca aga ccc agc gta aat gca gtg 912 Ile Ser Lys Leu Thr Ile Tyr Asp Ala Arg Pro Ser Val Asn Ala Val 290 295 300 gcc aac aag gca aca gga gga gga tat gaa agt gat gat gca tat cat 960 Ala Asn Lys Ala Thr Gly Gly Gly Tyr Glu Ser Asp Asp Ala Tyr His 305 310 315 320 aac gcc gaa ctt ttc ttc tta gac att cat aat att cat gtt atg cgg 1008 Asn Ala Glu Leu Phe Phe Leu Asp Ile His Asn Ile His Val Met Arg 325 330 335 gaa tct tta aaa aaa gtg aag gac att gtt tat cct aat gta gaa gaa 1056 Glu Ser Leu Lys Lys Val Lys Asp Ile Val Tyr Pro Asn Val Glu Glu 340 345 350 tct cat tgg ttg tcc agt ttg gag tct act cat tgg tta gaa cat atc 1104 Ser His Trp Leu Ser Ser Leu Glu Ser Thr His Trp Leu Glu His Ile 355 360 365 aag ctc gtt ttg aca gga gcc att caa gta gca gac aaa gtt tct tca 1152 Lys Leu Val Leu Thr Gly Ala Ile Gln Val Ala Asp Lys Val Ser Ser 370 375 380 ggg aag agt tca gtg ctt gtg cat tgc agt gac gga tgg gac agg act 1200 Gly Lys Ser Ser Val Leu Val His Cys Ser Asp Gly Trp Asp Arg Thr 385 390 395 400 gct cag ctg aca tcc ttg gcc atg ctg atg ttg gat agc ttc tat agg 1248 Ala Gln Leu Thr Ser Leu Ala Met Leu Met Leu Asp Ser Phe Tyr Arg 405 410 415 agc att gaa ggg ttc gaa ata ctg gta caa aaa gaa tgg ata agt ttt 1296 Ser Ile Glu Gly Phe Glu Ile Leu Val Gln Lys Glu Trp Ile Ser Phe 420 425 430 gga cat aaa ttt gca tct cga ata ggt cat ggt gat aaa aac cac acc 1344 Gly His Lys Phe Ala Ser Arg Ile Gly His Gly Asp Lys Asn His Thr 435 440 445 gat gct gac cgt tct cct att ttt ctc cag ttt att gat tgt gtg tgg 1392 Asp Ala Asp Arg Ser Pro Ile Phe Leu Gln Phe Ile Asp Cys Val Trp 450 455 460 caa atg tca aaa cag ttc cct aca gct ttt gaa ttc aat gaa caa ttt 1440 Gln Met Ser Lys Gln Phe Pro Thr Ala Phe Glu Phe Asn Glu Gln Phe 465 470 475 480 ttg att ata att ttg gat cat ctg tat agt tgc cga ttt ggt act ttc 1488 Leu Ile Ile Ile Leu Asp His Leu Tyr Ser Cys Arg Phe Gly Thr Phe 485 490 495 tta ttc aac tgt gaa tct gct cga gaa aga cag aag gtt aca gaa agg 1536 Leu Phe Asn Cys Glu Ser Ala Arg Glu Arg Gln Lys Val Thr Glu Arg 500 505 510 act gtt tct tta tgg tca ctg ata aac agt aat aaa gaa aaa ttc aaa 1584 Thr Val Ser Leu Trp Ser Leu Ile Asn Ser Asn Lys Glu Lys Phe Lys 515 520 525 aac ccc ttc tat act aaa gaa atc aat cga gtt tta tat cca gtt gcc 1632 Asn Pro Phe Tyr Thr Lys Glu Ile Asn Arg Val Leu Tyr Pro Val Ala 530 535 540 agt atg cgt cac ttg gaa ctc tgg gtg aat tac tac att aga tgg aac 1680 Ser Met Arg His Leu Glu Leu Trp Val Asn Tyr Tyr Ile Arg Trp Asn 545 550 555 560 ccc agg atc aag caa caa cag ccg aat cca gtg gag cag cgt tac atg 1728 Pro Arg Ile Lys Gln Gln Gln Pro Asn Pro Val Glu Gln Arg Tyr Met 565 570 575 gag ctc tta gcc tta cgc gac gaa tac ata aag cgg ctt gag gaa ctg 1776 Glu Leu Leu Ala Leu Arg Asp Glu Tyr Ile Lys Arg Leu Glu Glu Leu 580 585 590 cag ctc gcc aac tct gcc aag ctt tct gat ccc cca act tca cct tcc 1824 Gln Leu Ala Asn Ser Ala Lys Leu Ser Asp Pro Pro Thr Ser Pro Ser 595 600 605 agt cct tcg caa atg atg ccc cat gtg caa act cac ttc tgagggggga 1873 Ser Pro Ser Gln Met Met Pro His Val Gln Thr His Phe 610 615 620 ccctggcacc gcattagagc tcgaaataaa ggcgatagct gactttcatt tggggcattt 1933 gtaaaaagta gattaaaata tttgcctcca tgtagaactt gaactaacat aatcttaaac 1993 tcttgaatat gtgccttcta gaatacatat tacaagaaaa ctacagggtc cacacggcaa 2053 tcagaagaaa ggagctgaga tgaggttttg gaaaaccctg acacctttaa aaagcagttt 2113 ttgaaagaca aaatttagat ttaatttacg tcttgagaaa tactatatat acaatatata 2173 tggggggggc ttaattgaaa caacattatt ttaaaatcaa aggggatata tgtttgtgga 2233 tggattttcc tgaagctgca ttaacagttg ctttggattc tctaagatga atccaaatgt 2293 gaaagatgca tgttactgcc aaaaccaaat tgagctcagc ttcctaggca ttacccaaaa 2353 gcaaggtgtt taagtaattg ccagctttta taccatcatg agtggtgact taaggagaaa 2413 tagctgtata gatgagtttt tcattatttg gaaatttagg ggtagaaaat gttttcccct 2473 aattttccag agaagcctat ttttatattt ttaaaaaact gacagggccc agttaaatat 2533 gatttgcatt ttttaaattt gccagtttta ttttctaaat tctttcatga gcttgcctaa 2593 aattcggaat ggttttcggg ttgtggcaaa ccccaaagag agcactgtcc aaggatgtcg 2653 ggagcatcct gctgcttagg ggaatgtttt cgcaaatgtt gctctagtca gtccagctca 2713 tctgccaaaa tgtagggcta ccgtcttgga tgcatgagct attgctagag catcatcctt 2773 agaaatcagt gccccagatg tacatgtgtt gagcgtattc ttgaagtatt gtgtttatgc 2833 atttcaattt caatggtgtt ggcttcccct ccccacccca cgcgtgcata aaaactggtt 2893 ctacaaattt ttacttgaag taccaggccg tttgcttttt caggttgttt tgttttatag 2953 tattaagtga aattttaaat gcacagttct atttgctatc tgaactaatt catttattaa 3013 gtatatttgt aaaagctaag gctcgagtta aaacaatgaa gtgttttaca atgatttgta 3073 aaggactatt tataactaat atggttttgt tttcaatgaa ttaagaaaga ttaaatatat 3133 ctttgtaaat tattttatgt catagttaat tggtctccca agtaagacat ctcaaataca 3193 gtagtataat gtatgaattt tgtaagtata agaaatttta ttagacattc tcttactttt 3253 tgtaaatgct gtaaatattt cataaattaa caaagtgtca ctccataaaa agaaagctaa 3313 tactaatagc ctaaaagatt ttgtgaaatt tcatgaaaac tttttaatgg caataatgac 3373 taaagacctg ctgtaataaa tgtattaact gaaacctaaa aaaaaaaaaa aaaaaaaa 3431 2 621 PRT German-type microorganism & cell cul. 2 Ala Ala Glu Gln Pro Gly Asn Gly Gly Gly Ala Arg Ser Pro Arg Val 1 5 10 15 Ser Arg Met Ala Ser Ala Ser Thr Ser Lys Tyr Asn Ser His Ser Leu 20 25 30 Glu Asn Glu Ser Ile Lys Arg Thr Ser Arg Asp Gly Val Asn Arg Asp 35 40 45 Leu Thr Glu Ala Val Pro Arg Leu Pro Gly Glu Thr Leu Ile Thr Asp 50 55 60 Lys Glu Val Ile Tyr Ile Cys Pro Phe Asn Gly Pro Ile Lys Gly Arg 65 70 75 80 Val Tyr Ile Thr Asn Tyr Arg Leu Tyr Leu Arg Ser Leu Glu Thr Asp 85 90 95 Ser Ser Leu Ile Leu Asp Val Pro Leu Gly Val Ile Ser Arg Ile Glu 100 105 110 Lys Met Gly Gly Ala Thr Ser Arg Gly Glu Asn Ser Tyr Gly Leu Asp 115 120 125 Ile Thr Cys Lys Asp Met Arg Asn Leu Arg Phe Ala Leu Lys Gln Glu 130 135 140 Gly His Ser Arg Arg Asp Met Phe Glu Ile Leu Thr Arg Tyr Ala Phe 145 150 155 160 Pro Leu Ala His Ser Leu Pro Leu Phe Ala Phe Leu Asn Glu Glu Lys 165 170 175 Phe Asn Val Asp Gly Trp Thr Val Tyr Asn Pro Val Glu Glu Tyr Arg 180 185 190 Arg Gln Gly Leu Pro Asn His His Trp Arg Ile Thr Phe Ile Asn Lys 195 200 205 Cys Tyr Glu Leu Cys Asp Thr Tyr Pro Ala Leu Leu Val Val Pro Tyr 210 215 220 Arg Ala Ser Asp Asp Asp Leu Arg Arg Val Ala Thr Phe Arg Ser Arg 225 230 235 240 Asn Arg Ile Pro Val Leu Ser Trp Ile His Pro Glu Asn Lys Thr Val 245 250 255 Ile Val Arg Cys Ser Gln Pro Leu Val Gly Met Ser Gly Lys Arg Asn 260 265 270 Lys Asp Asp Glu Lys Tyr Leu Asp Val Ile Arg Glu Thr Asn Lys Gln 275 280 285 Ile Ser Lys Leu Thr Ile Tyr Asp Ala Arg Pro Ser Val Asn Ala Val 290 295 300 Ala Asn Lys Ala Thr Gly Gly Gly Tyr Glu Ser Asp Asp Ala Tyr His 305 310 315 320 Asn Ala Glu Leu Phe Phe Leu Asp Ile His Asn Ile His Val Met Arg 325 330 335 Glu Ser Leu Lys Lys Val Lys Asp Ile Val Tyr Pro Asn Val Glu Glu 340 345 350 Ser His Trp Leu Ser Ser Leu Glu Ser Thr His Trp Leu Glu His Ile 355 360 365 Lys Leu Val Leu Thr Gly Ala Ile Gln Val Ala Asp Lys Val Ser Ser 370 375 380 Gly Lys Ser Ser Val Leu Val His Cys Ser Asp Gly Trp Asp Arg Thr 385 390 395 400 Ala Gln Leu Thr Ser Leu Ala Met Leu Met Leu Asp Ser Phe Tyr Arg 405 410 415 Ser Ile Glu Gly Phe Glu Ile Leu Val Gln Lys Glu Trp Ile Ser Phe 420 425 430 Gly His Lys Phe Ala Ser Arg Ile Gly His Gly Asp Lys Asn His Thr 435 440 445 Asp Ala Asp Arg Ser Pro Ile Phe Leu Gln Phe Ile Asp Cys Val Trp 450 455 460 Gln Met Ser Lys Gln Phe Pro Thr Ala Phe Glu Phe Asn Glu Gln Phe 465 470 475 480 Leu Ile Ile Ile Leu Asp His Leu Tyr Ser Cys Arg Phe Gly Thr Phe 485 490 495 Leu Phe Asn Cys Glu Ser Ala Arg Glu Arg Gln Lys Val Thr Glu Arg 500 505 510 Thr Val Ser Leu Trp Ser Leu Ile Asn Ser Asn Lys Glu Lys Phe Lys 515 520 525 Asn Pro Phe Tyr Thr Lys Glu Ile Asn Arg Val Leu Tyr Pro Val Ala 530 535 540 Ser Met Arg His Leu Glu Leu Trp Val Asn Tyr Tyr Ile Arg Trp Asn 545 550 555 560 Pro Arg Ile Lys Gln Gln Gln Pro Asn Pro Val Glu Gln Arg Tyr Met 565 570 575 Glu Leu Leu Ala Leu Arg Asp Glu Tyr Ile Lys Arg Leu Glu Glu Leu 580 585 590 Gln Leu Ala Asn Ser Ala Lys Leu Ser Asp Pro Pro Thr Ser Pro Ser 595 600 605 Ser Pro Ser Gln Met Met Pro His Val Gln Thr His Phe 610 615 620 3 34 DNA German-type microorganism & cell cul. 3 cagggatccg atggcagccg agcagcctgg caac 34 4 33 DNA German-type microorganism & cell cul. 4 gggggatcct cagaagtgag tttgcacatg ggg 33 

What is claimed is:
 1. A purified tyrosine phosphatase-related protein, comprising the amino acid sequence of FIG. 1 (SEQ ID NO:2) or an amino acid sequence differing therefrom by one or several amino acids, wherein the DNA encoding the latter amino acid sequence hybridizes completely with tie DNA of FIG. 1 (SEQ ID NO:1) at 20° C. below the melting point of the DNA, and wherein the protein disturbs the differentiation of muscle cells.
 2. An isolated DNA encoding the protein according to claim 1, wherein the DNA comprises; (a) the DNA of FIG. 1 (SEQ ID NO:1) or a DNA differing therefrom by one or several base pairs, the latter DNA completely hybridizing with the DNA of FIG. 1 (SEQ ID NO:1) at 20° C. below the melting point of the DNA, or b) a DNA related to the DNA of (a) via the degeneracy of the genetic code.
 3. An expression plasmid, comprising the DNA according to claim
 2. 4. A transformant comprising the expression plasmid according to claim
 3. 5. A process for the preparation of the protein according to claim 1, wherein said process comprises culturing a transformant containing a expression plasmid comprising: (a) the DNA of FIG. 1 (SEQ ID NO:1) or a DNA differing therefrom by one or several base pairs, the latter DNA completely hybridizing with the DNA of FIG. 1 (SEQ ID NO:1) at 20° C. below the melting point of the DNA, or (b) a DNA related to the DNA of (a) via the degeneracy of the genetic code.
 6. A reagent for treating disturbed cell differentiation comprising the protein according to claim
 1. 7. The reagent according to claim 6, wherein said disturbed cell differentiation is myotubular myopathy.
 8. A reagent for the diagnosis and/or treatment of disturbed cell differentiation comprising the DNA according to claim
 2. 9. The reagent according to claim 8, wherein said disturbed cell differentiation is myotubular myopathy. 